Enzymatic Properties of Recombinant Ligase Butelase-1 and Its Application in Cyclizing Food-Derived Angiotensin I-Converting Enzyme Inhibitory Peptides

Abstract

Butelase-1 is an efficient ligase from Clitoria ternatea with wide applications in the food and biopharmaceutical fields. This research aimed to achieve high-efficiency expression of butelase-1 and explore its application in food-derived angiotensin I-converting enzyme (ACE) inhibitory peptides. The recombinant butelase-1 zymogen was prepared at a yield of 100 mg/L in Escherichia coli and successfully activated at pH 4.5, resulting in a 6973.8 U/L yield of activated butelase-1 with a specific activity of 348.69 U/mg and a catalytic efficiency of 9956 M–1 s–1. Activated butelase-1 exhibited considerable resistance to Tween-20, Triton X-100, and methanol. The “traceless” cyclization of ACE inhibitory peptides was realized using activated butelase-1, which resulted in higher stability and ACE inhibitory activity than those of the linear peptides. Our work proposed an efficient method for the preparation of butelase-1 and provided a promising model for its application in food fields.

Butelase 1-Mediated Enzymatic Cyclization of Antimicrobial Peptides: Improvements on Stability and Bioactivity

“…Our previous studies have shown that recombinant butelase 1 exhibited maximum cyclization activity at 45 °C . Here, the cyclization reaction was performed at 40 °C to alleviate the effect of heating on the activity of AMPs.…”

“…Butelase 1 was prepared according to our previous report with a brief modification. 23 In brief, the recombinant vector pMAL-c6t-butelase 1 was transformed into the E. coli cell. The cells were grown in LB medium at 37 °C until the absorbance of culture reached 0.4−0.6 at 600 nm, and then, 0.1 mM isopropyl β-D-thiogalactoside was added to the culture for inducing target protein.…”

“…The thermostability and anti-protease hydrolyzation of AMPs were also investigated. Solutions containing 0.25 mg/mL AMPs were heated at different temperatures (37, 47, 57, 67, 77, 87, and 97 °C) for 1 h to analyze the thermal stability of the peptides . The peptides remaining after heating were calculated based on the RP-HPLC profile.…”

“…Butelase 1 was prepared according to our previous report with a brief modification . In brief, the recombinant vector pMAL-c6t-butelase 1 was transformed into the E.…”

Consensus design and engineering of an efficient and high-yield Peptide Asparaginyl Ligase

“…Recombinant PAL proenzymes can be expressed in bacteria, but the expressed proteins are often present as misfolded proteins in inclusion bodies (Nguyen et al, 2014) and the yield of soluble proteins is fairly low (Tang & Luk, 2021). Although incorporation of large fusion tags such as MBP can increase the yield of crude fusion proteins, they can also lead to heterogeneous activity of the enzymes after activation (Zhao et al, 2021; Zhao et al, 2022). Secretory expression in insect cells could produce about 20 mg/L of VyPAL2, but this approach is limited by relatively high costs.…”